1. Field of the Invention
The invention relates to the detection and measurement of predetermined immunochemical substances, using a novel method and apparatus therefor which provide for rapid qualitative and quantitative determinations in an advantageous manner.
2. Description of the Prior Art and Other Information
Various methods have been developed in the last two or three decades for the determination of a variety of immunochemical substances, including antigens, antibodies, haptens, and certain low molecular weight substances. Examples of these methods are:
1. Radioassay techniques PA1 2. Fluoroimmunoassays (FIA) PA1 3. Enzyme immunoassay (EIA) PA1 4. Lysis-initiating immunoassays (LIA) PA1 5. Latex-particle agglutination (LPA) PA1 6. Charcoal-particle agglutination (CPA) PA1 7. Hemagglutination and Hemagglutination Inhibition Assays (HA), (HIA) PA1 8. Complement Fixation (CF) PA1 9. Counter-immunoelectrophoresis (CIEP) PA1 10. Radial Immunodiffusion and Double diffusion (RID) PA1 11. Viroimmunoassay (VIA); and PA1 12. Spin immunoassay (SIA) among others. PA1 13. Turbidity (physical assay)
a. Competitive protein binding assays PA2 b. Radioimmunoassay (RIA) PA2 c. Immunoradiometric assays PA2 d. Sandwich or 2-site immunoradiometric assays
One type of immunochemical test system involves the use of labels. Within these, there are many types of labels useful in assays for the detection and measurement in serum or other media of biologically important or interesting compounds or substances. The administration of most of these tests are hampered by one or more of the following limitations: (1) lack of sensitivity, (2) complexity of the test procedure, (3) instability of reagents, (4) hazardous reagents, (5) impure reagents, and (6) expensive equipment required to perform quantitative and qualitative analysis of the amount of label involved in an immunochemical reaction. For a review of the development and evaluation of immunological methods and their uses as diagnostic tools, reference is made to "Immunology as a Laboratory Tool", by FRANZ PEETOOME, 37 American Journal of Technology (2) 445-469 (1971), incorporated herein.
There are other immunochemical test systems which do not use labels for a means of detection; some of these are the so-called "agglutination" tests, wherein the analysis depends on the measurement of certain electromagnetic radiation properties of the liquid samples containing immunochemical constituents, with the measured properties depending on whether or not an immunochemical reaction has taken place. A pioneer reference in this area of technology is Schuurs, U.S. Pat. No. 3,551,555 (1970). See also Price et al, U.S. Pat. No. 4,066,744 (1978); both patents are incorporated herein by reference. Examples of these "agglutination" tests include the so-called "NOSTICON" latex tests by Organon Incorporated (West Orange, New Jersey), including PREGNOSTICON.RTM., RHEUMANOSTICON.RTM., and GONOSTICON.RTM. latex agglutination or latex agglutination inhibition Slide Tests (distinguishable from the "NOSTICON" erythrocyte agglutination inhibition tests).
It must be emphasized that both labeled and unlabeled immunochemical testing may employ various devices to separate immunochemical constituents which have reacted from non-reacted immunochemical constituents and from substances irrelevant to the test. For example, some EIA patents require separation through the use of one component in the antigen-antibody reaction being in an "insolubilized" phase for separation-see Schuurs and co-workers in U.S. Pat. Nos. 3,654,090; 3,791,932; 3,850,752; 3,839,153; 3,879,262; 4,016,043 and Reissue 29,169.
Another method does not require separation of free and bound label because the assay depends on the inhibition or activation of the enzyme label by antibody binding (e.g., the EMIT.RTM.-type system of Syva Corporation of Palo Alto, California, for EIA and FRAT, or "free radical assay technique", for SIA)-see U.S. Pat. Nos. 3,880,715; 3,852,157; 3,875,011; 3,935,074; and 3,905,871, and an article by Kenneth S. Rubenstein et al in "Homogenous Enzyme Immunoassay, a New Immunochemical Technique", Biochemical and Biophysical Research Communications 47, No. 4, 846-851 (1972) (all incorporated herein by reference). These are examples wherein an insolubilized phase is not employed and the assay depends on inhibition or activation of the enzyme label by antibody binding. See also G. Brian Wisdom, "Enzyme Immunoassay", Clinical Chemistry 22/8, 1243 (1976).
Radioimmunoassay (RIA) is now considered a classical and well-known technique for detecting antigens at very low concentrations. It is based upon the competition between radio-labeled and unlabeled antigen for a fixed, limited amount of antibody, as described by R. Yalow and S. Berson in J. Clin. Invest., 39 1157 (1960). The amount of unlabeled antigen influences the distribution of the labeled antigen in antibody bound (B) and antibody-free (F) labeled antigen, i.e., the more that unlabeled antigen is present, the less the labeled antigen is able to combine with the antibody. In order to obtain conclusive results from the distribution, a good separation between B and F must be made. Methods used for this purpose are, for instance, chromatoelectrophoresis, described by S. Berson and R. Yalow in The Hormones, edited by G. Pincus et al, Academic Press, New York (1964), vol. IV, 557, or insolubilization of the antibodies. This insolubilization can be achieved by chemical means (cross-linking or covalent binding to an insoluble carrier) or by physical methods (adsorption to an insoluble carrier).
Of the limitations cited above, a most serious limitation until recently has been lack of adequate sensitivity to detect some antigens. In general, three levels of sensitivity are recognizable. Low sensitivity techniques, where materials detected and measured exist in microgram/milliliter quantities, include RID, CF, CEP, CPA, and LPA. Intermediate sensitivity techniques, where microgram/milliliter to nanogram/milliliter quantities of materials may be measured, include HIA, HA, CF, FIA, SIA, VIA, and EIA. Until recently only RIA was able to measure with ultrasensitivity the picogram/milliliter to femtogram/milliliter region.
Many of the techniques listed above require that some form of physically or chemically identifiable label be attached to reagents in the assay system in order that the result of a test can be detected. RIA, FIA, EIA, LIA, VIA, and SIA all fall into this category. Radioactivity, fluorescent moieties, enzymes, complement, viruses, and electron-spin labels are used respectively to generate some form of end-point signal. The sensitivity with which these labels can be detected directly and fundamentally affects the useful ranges of the test systems using them.
The sensitivity with which a labeling moiety can be measured depends upon the nature of the signal that it generates, the ability to detect that signal, and the intensity of signal available per unit amount of marker molecule, i.e., its specific activity. With radioactive labels, heretofore the most popular label in use, the signal is decay radiation. Because of the penetrating properties of the emissions generated, radioactive decay can be detected easily. Modern counting equipment very efficiently measures the radioactive emissions from even a small amount of radioactive material. Furthermore, there is a range of specific activities offered by isotopes currently used for tagging.
As noted, up to the present time, the radioimmunoassay (RIA) method in its various forms has been the most sensitive system available. The RIA method, unfortunately, has several serious disadvantages, including the requirement of special equipment, trained staff, the recited need for extra safety measures to protect against harmful radiation, special licensing, controlled radioactive wastes disposal and the continuous disappearance of labeled compound by radioactive decay. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 in an article by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodies and Immunological Assay Systems", Lancet, II, 492 (1968), and Nature 219, 168 (1968). No procedural details were provided, the article offered only the general idea, leaving it to future workers to determine the basic step and to perform the extensive experimentation needed to establish a practical operative enzymatic immunoassay method.
More recently, methods for detecting and measuring immunochemical substances have been developed in which, in lieu of a radioactive isotope, immunochemical substances have been labeled with other materials which can be detected by various techniques, e.g., optical and electronic instrument methods. One useful group of materials are enzymes which, because of the great number of analytical permutations, has created a whole family of techniques known collectively as enzyme immunoassay (EIA) techniques.
Among the more recent patents issued that are representative of the state of the art in the detection and measurement of immunochemical substances are, as recited, U.S. Pat. Nos. 3,654,090, issued Apr. 4, 1972; 3,666,421, issued May 30, 1972; 3,791,932, issued Feb. 12, 1974; 3,839,153, issued Oct. 1, 1974; 3,850,752, issued Nov. 26, 1974; 3,879,262, issued Apr. 22, 1975; 4,016,043, issued Apr. 5, 1977; and Reissue Pat. No. 29,169, reissued Apr. 5, 1977, all incorporated by reference herein.
A specific example of a recent latex agglutination inhibition method is a qualitative in vitro test for determining the presence of human chorionic gonadotropin (HCG). HCG is a hormone that is characteristic of pregnancy and may be found in the urine of a pregnant human. An antiserum specific to HCG can be prepared from rabbits immunized with HCG to produce the antibody.
According to the PREGNOSTICON.RTM. Slide Test, if the antiserum is mixed with latex that has been sensitized with HCG, agglutination of the latex occurs. If, on the other hand, the antiserum is mixed with a sample of urine containing HCG, i.e., from a pregnant person, the antiserum is neutralized, and upon subsequent mixing of the antiserum-urine mixture with the HCG-sensitized latex, the agglutination of the latex is inhibited. The latex appears as a milky homogenous suspension, its agglutination having been inhibited. This is a positive test for pregnancy.
Although usually a positive or negative result can be determined by of lack of agglutination or agglutination of the latex, respectively, a maximum inhibition of agglutination may not occur in the early stages of pregnancy when the concentration of HCG in the urine has not increased above a certain threshold level which can be detected by this method. The sensitivity of the described pregnancy test is normally such that the concentration levels of HCG are usually sufficiently elevated by the twelfth day after menstruation fails to occur that the HCG can be detected with the test. If the result of the test is inconclusive, the test must be conducted again in another week or two, to allow sufficient time for an increase in the HCG concentration in the urine to detectable levels. Of course, this is very undesirable from a diagnostic viewpoint, since it is often important to be able to determine the existence of pregnancy at the earliest stages.
The above-described test is qualitative in nature, giving either a positive or a negative test for some threshold concentration of HCG. If HCG is detected in a urine sample, a more quantitative determination of the concentration of HCG in the sample can be made by conducting a series of tests on a series of systematic serial dilutions (commonly referred to in the art as a "dilution series") of the urine samples. Of course, the necessity of conducting a series of tests to determine the concentration of HCG in a single urine sample is time-consuming and costly.
In an available embodiment, the foregoing technique for qualitatively detecting the HCG antigen characteristic of pregnancy is known as the PREGNOSTICON.RTM.-Slide Test (Organon Inc., West Orange, New Jersey).
The same general agglutination process principles underlying the PREGNOSTICON.RTM.-Slide Test (and erythrocyte test) can also be applied to the determination of other immunochemical substances which can be specifically bound, such as antigens i.e., those associated with gonorrhea, rheumatoid factor, etc., and antibodies such as those specific to GC, IgG, and so forth. It would be desirable to provide to provide a test for determining immunochemical substances which would both detect and quantitatively measure the presence of such specifically-bindable immunochemical substances rapidly and at low concentrations, to thereby insure early diagnosis.
The use of light-scattering photometers in analyzing the electromagnetic properties of various substances is well known and photometric methods can be used in the analysis of immunochemical substances. There are many different embodiments in which light-scattering photometers have been used, but most cases, such instrumentation is very sophisticated and expensive. One reason for this is that such instruments typically include a number of lens systems and complicated mechanisms for positioning the cuvette containing the substance being determined, for example, the BRICE-PHOENIX Model OM-2000 Light Scattering Photometer (Virtis Co., Gardiner, New York); SCIENCE SPECTRUM Differential Light-Scattering.RTM. Photometer (Santa Barbara, Cal.). For example, U.S. Pat. No. 3,036,492 issued May 29, 1962, describes a complex adjustable specimen chamber for determining the light transmittance properties of a sample at varying angles. Another example is U.S. Pat. No. 3,918,817 issued Nov. 11, 1975, which describes a turbidimeter, a particular type of photometer, including a special thermal insulating housing and using a glass test tube of rectangular cross-section. Buffone, "Improved Nephelometric Instrumentation", Laboratory Management, April, 1977, describes at page 19 a nephelometer, a similar type of photometer, using an incandescent lamp with filters to produce a band of radiation between 450 and 650 mn.
In general, much of the existing literature has been concerned primarily with textile quality control techniques. For example, in other variations involving the use of light-scattering photometers, textile color analyzers involving instrument heads using a plurality of fiber-optic bundles positioned to receive diffuse light reflected from the textile samples have been devised, as described in U.S. Pat. No. 3,986,778 issued Oct. 19, 1976, and U.S. Pat. No. 3,999,860 issued Dec. 28, 1976.
It is interesting to note that light-scattering photometric methods for determining particular substances have in the past typically required measurement at a particular wavelength, that is, essentially monochromatic light.
For instance, in Blume and Greenberg, "Application of Differential Light Scattering to the Latex Agglutination Assay for Rheumatoid Factor" Clinical Chemistry, Vol. 21, No. 9, 1975, page 1234 et seq., it is disclosed that in the technique of differential light scattering it is essential that the light source be highly monochromatic, such as, e.g., that produced by a helium/neon-laser (632.8 nm). The requirement of essentially monochromatic light in photometric determinations is again described by Lichenbelt, Pallmarnanobaran, and Wiersema, "Rapid Coagulation of Polystyrene Latex in a Stopped Flow Spectrophotometer", Journal of Colloid & Interface Science, Vol. 49, No. 2, 1974, page 281 et seq., and Dininno and McCandless, "Agarose Medium Turbidimetric Assay for Cross-Reacting Antigens", Journal of Immunological Methods, Vol. 17, 1977, pages 73-79. See also Flurometry Reviews, March 1969 (monthly bulletin of Turner Inc., Palo Alto, California).
Surprisingly, it has now been found in the instant invention that for the detection and measurement of insolubilized particles in suspension, i.e., non-agglutinated or agglutinated latex particles, turbid liquid samples, chemical precipitates, etc., the requirement for a monochromatic, incident light source is illusory. It has also been found in the instant invention that the widest spectral band of incident light available, whose upper wavelength limit being equal to or less than the mean diameter of the insolubilized particles in the suspension of interest (the value herein which may be expressed in nanometers or microns), is preferred for optimum detection and measurement sensitivity. The use of such wide-band spectral filters, commonly known as low-pass optical filters, in association with an appropriate light source, is indeed unique and novel for the aforementioned applications.
The use of such low-pass optical filters are suitable for forward-scattering measurements at the 0.degree. optical-axis mode as well as for light-scattering measurements at any off-axis detection angle mode from 1.degree. to 90.degree. to the optical axis of the text system. The 90.degree. detection mode is more commonly known as the nephelometric mode. Examples of present art for the 0.degree. detection mode is the Klett-Summerson Model 900-3 colorimeter (Klett Mfg. Co.); for off-axis detection modes, the Brice-Phoenix Models 2000D and 2000DM Light-Scattering Photometers (Phoenix Precision Instrument Company); and for the 90.degree. detection mode, the Volu-Sol Models 300 and 299 Nephelometers (Volu-Sol Medical Ind., Inc.).
The preceding discussion illustrates that a need exists for (a) new improved methods and (b) low cost, simple to use apparatus therefor for making rapid, accurate, and economical measurements of immunochemical substances in agglutination and colorimetric medical-diagnostic laboratory tests employing inexpensive disposable cuvettes (having scratches and small defects) normally manufactured for the science laboratory.